We and others have demonstrated that silencing of tumor suppressor genes associated with promoter hypermethylation is a common feature of many human cancers. Loss of p16 activity through hypermethylation of the promoter is associated with loss of the expression and function early in the progression of head and neck cancer. Methylation of p16 and other selected methylated markers have been identified in the saliva and serum of patients with head and neck cancer. We propose to thoroughly catalog hypermethylated genes in squamous cell carcinoma of the head neck (HNSCC) and will identify novel hypermethylated genes by: (1). A candidate gene approach based on testing genes found to be hypermethylated in other tumors or on the functional structure and biologic plausibility of the candidate gene and (2) A functional screen looking for genes that are up-regulated following treatment with demethylating agents (e.g.5-aza-deoxycytidine). Newly identified hypermethylated genes will be characterized in tumor progression. Moreover, these genes will be tested as markers in cancer detection. Optimization of sensitive real-time PCR assays able to detect specific methylation will allow us to test these markers in paired saliva and serum samples from patients with head and neck cancer for initial analytical validation. A screening study in the larger SPORE proposal (project #1) will allow us to further test these markers and determine the feasibility of this approach for the early detection and monitoring of patients with head and neck cancer.